Bottom Line:
Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex.The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them.It is likely that the knowledge of the genomes will be largely transferable between species within the complex.

Background: The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf fescues, both belonging to the subfamily Pooideae. This subfamily also includes wheat, barley, oat and rye, making it extremely important to world agriculture. Species within the Lolium-Festuca complex show very diverse phenotypes, and many of them are related to agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity.

Results: We have generated de novo transcriptome assemblies for four species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some of the differences between the more stress tolerant Festuca, and the less stress tolerant Lolium species.

Conclusions: Our data presents a comprehensive transcriptome sequence comparison between species from the Lolium-Festuca complex, with the identification of potential candidate genes underlying some important phenotypical differences within the complex (such as VRN2). The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.

Fig6: Organisation of proteins under positive selection inFestuca toLoliums comparison. The diagram show the number of proteins under positive selection between Festuca and analyzed Lolium species.

Mentions:
The number of orthologous pairs for which Ka and Ks could be calculated and was above 1 was equal to: 210 for F. pratensis and L. multiflorum (Additional file 7: Table S7), 177 for F. pratensis and L. m. westerwoldicum (Additional file 8: Table S8), 203 for F. pratensis and L. temulentum (Additional file 9: Table S9), 124 for F. pratensis and L. perenne (Additional file 10: Table S10). All of the pairs have been linked to their functional annotations. We have then categorized the transcript pairs that are under positive pressure in multiple comparisons, by checking how many Festuca identifiers are being shared between the pairwise comparisons (Figure 6). The majority of pairs have shown Ka/Ks values over 1 in only a single pair-wise comparison. However, there were three pairs shared in every analyzed comparison, and thus differentiating the Festuca from the Lolium species. The first was a homologue of A. thaliana ribosomal protein L4, one of the primary rRNA binding proteins [62], and the second was a UNC93-like protein 2, which is an integral component of the cell membrane [63]. In addition, in every pairwise comparison we detected a homologue of disease resistance protein RPM1, involved in plant defense against P. syringae in A. thaliana [64]. A homologue to disease resistance protein RPP13 conferring resistance to Peronospora parasitica in A. thaliana [65] has been identified in L. perenne, L. multiflorum and L. temulentum comparisons. Other proteins involved in plant-pathogen interaction, RPP8 and RPH8A, have been found in the L. multiflorum comparison. Different types of F-box proteins, which are mediating protein-protein interactions, were also abundant in every pairwise comparison.Figure 6

Fig6: Organisation of proteins under positive selection inFestuca toLoliums comparison. The diagram show the number of proteins under positive selection between Festuca and analyzed Lolium species.

Mentions:
The number of orthologous pairs for which Ka and Ks could be calculated and was above 1 was equal to: 210 for F. pratensis and L. multiflorum (Additional file 7: Table S7), 177 for F. pratensis and L. m. westerwoldicum (Additional file 8: Table S8), 203 for F. pratensis and L. temulentum (Additional file 9: Table S9), 124 for F. pratensis and L. perenne (Additional file 10: Table S10). All of the pairs have been linked to their functional annotations. We have then categorized the transcript pairs that are under positive pressure in multiple comparisons, by checking how many Festuca identifiers are being shared between the pairwise comparisons (Figure 6). The majority of pairs have shown Ka/Ks values over 1 in only a single pair-wise comparison. However, there were three pairs shared in every analyzed comparison, and thus differentiating the Festuca from the Lolium species. The first was a homologue of A. thaliana ribosomal protein L4, one of the primary rRNA binding proteins [62], and the second was a UNC93-like protein 2, which is an integral component of the cell membrane [63]. In addition, in every pairwise comparison we detected a homologue of disease resistance protein RPM1, involved in plant defense against P. syringae in A. thaliana [64]. A homologue to disease resistance protein RPP13 conferring resistance to Peronospora parasitica in A. thaliana [65] has been identified in L. perenne, L. multiflorum and L. temulentum comparisons. Other proteins involved in plant-pathogen interaction, RPP8 and RPH8A, have been found in the L. multiflorum comparison. Different types of F-box proteins, which are mediating protein-protein interactions, were also abundant in every pairwise comparison.Figure 6

Bottom Line:
Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex.The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them.It is likely that the knowledge of the genomes will be largely transferable between species within the complex.

Background: The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf fescues, both belonging to the subfamily Pooideae. This subfamily also includes wheat, barley, oat and rye, making it extremely important to world agriculture. Species within the Lolium-Festuca complex show very diverse phenotypes, and many of them are related to agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity.

Results: We have generated de novo transcriptome assemblies for four species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some of the differences between the more stress tolerant Festuca, and the less stress tolerant Lolium species.

Conclusions: Our data presents a comprehensive transcriptome sequence comparison between species from the Lolium-Festuca complex, with the identification of potential candidate genes underlying some important phenotypical differences within the complex (such as VRN2). The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.